title: Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells.
abstract: In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressin-induced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin-2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase.
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 10051, Taiwan.
authors.forename: Chin-San
authors.initials: CS
authors.lastname: Loo
authors.forename: Cheng-Wei
authors.initials: CW
authors.lastname: Chen
authors.forename: Po-Jen
authors.initials: PJ
authors.lastname: Wang
authors.forename: Pei-Yu
authors.initials: PY
authors.lastname: Chen
authors.forename: Shu-Yu
authors.initials: SY
authors.lastname: Lin
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.forename: Ming-Jiun
authors.initials: MJ
authors.lastname: Yu
mesh_terms: D000199:Actins / Q000378:metabolism*; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D050034:Antidiuretic Agents / Q000494:pharmacology*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D020543:Proteome / Q000378:metabolism*; D014667:Vasopressins / Q000494:pharmacology*; D014867:Water / Q000378:metabolism
chemical_list: D000199:Actins; D000276:Adjuvants, Immunologic; D050034:Antidiuretic Agents; D008840:Microfilament Proteins; D020543:Proteome; D014867:Water; D014667:Vasopressins; D005576:Colforsin
keywords: AQP2; LC-MS/MS; SILAC; dDAVP; proteome